Nonalcoholic fatty liver disease is the most common chronic liver disorder in developed countries. Its pathogenesis is poorly understood, and therapeutic options are limited. Here we show that SIRT7, an NAD+-dependent H3K18Ac deacetylase, functions at chromatin to suppress ER stress and prevents the development of fatty liver disease. SIRT7 is induced upon ER stress and is stabilized at the promoters of ribosomal proteins through its interaction with the transcription factor Myc to silence gene expression and to relieve ER stress. SIRT7 deficient mice develop chronic hepatosteatosis resembling human fatty liver disease. Myc inactivation or pharmacological suppression of ER stress alleviates fatty liver caused by SIRT7 deficiency. Importantly, SIRT7 suppresses ER stress and reverts the fatty liver disease in diet-induced obese mice. Our study identifies SIRT7 as a cofactor of Myc for transcriptional repression and delineates a druggable regulatory branch of the ER stress response that prevents and reverts fatty liver disease.

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc–Brn1b and linc–Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr−/− neonates, whereas linc–Brn1b−/− mutants displayed distinct abnormalities in the generation of upper layer II–IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules.

DOI:
http://dx.doi.org/10.7554/eLife.01749.001

eLife digest

The mammalian genome is comprised of DNA sequences that contain the templates for proteins, and other DNA sequences that do not code for proteins. The coding DNA sequences are transcribed to make messenger RNA molecules, which are then translated to make proteins. Researchers have known for many years that some of the noncoding DNA sequences are also transcribed to make other types of RNA molecules, such as transfer and ribosomal RNA. However, the true breadth and diversity of the roles played by these other RNA molecules have only recently begun to be fully appreciated.

Mammalian genomes contain thousands of noncoding DNA sequences that are transcribed. Recent in vitro studies suggest that the resulting long noncoding RNA molecules can act as regulators of transcription, translation, and cell cycle. In vitro studies also suggest that these long noncoding RNA molecules may play a role in mammalian development and disease. Yet few in vivo studies have been performed to support or confirm such hypotheses.

Now Sauvageau et al. have developed several lines of knockout mice to investigate a subset of noncoding RNA molecules known as long intergenic noncoding RNAs (lincRNAs). These experiments reveal that lincRNAs have a strong influence on the overall viability of mice, and also on a number of developmental processes, including the development of lungs and the cerebral cortex.

Given that the vast majority of the human genome is transcribed, the mouse models developed by Sauvageau et al. represent an important step in determining the physiological relevance, on a genetic level, of the noncoding portion of the genome in vivo.

Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide–binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A– or Ric-8B–deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A−/− ES cells, which resulted from reduced steady-state amounts of Gαi, Gαq, and Gα13 proteins to <5% of those of wild-type cells. The amounts of Gαs and total Gβ protein were partially reduced in Ric-8A−/− cells compared to those in wild-type cells, and only the amount of Gαs was reduced substantially in Ric-8B−/− cells. The abundances of mRNAs encoding the G protein α subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Gαi and Gαq were efficiently translated in Ric-8A−/− cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gβγ. Finally, Gαi, Gαq, and Gβ1 proteins exhibited accelerated rates of degradation in Ric-8A−/− cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Gα subunits with cellular membranes.

Skin wound repair requires complex and highly coordinated interactions between keratinocytes, fibroblasts and immune cells to restore the epidermal barrier and tissue architecture after acute injury. The cytokine interleukin-22 (IL-22) mediates unidirectional signaling from immune cells to epithelial cells during injury of peripheral tissues such as the liver and colon, where IL-22 causes epithelial cells to produce anti-bacterial proteins, express mucins, and enhance epithelial regeneration. In this study, we use IL-22−/− mice to investigate the in vivo role for IL-22 in acute skin wounding. We find that IL-22−/− mice display major defects in the skin’s dermal compartment after full thickness wounding. We find that IL-22 signaling is active in fibroblasts using in vitro assays with primary fibroblasts and that IL-22 directs extracellular matrix (ECM) gene expression as well as myofibroblast differentiation both in vitro and in vivo. These data define roles of IL-22 beyond epithelial crosstalk, and suggest that IL-22 plays a previously unidentified role in skin repair by mediating interactions between immune cells and fibroblasts.

Vascular endothelial growth factor (VEGF) is a key upstream mediator of tumor angiogenesis, and blockade of VEGF can inhibit tumor angiogenesis and decrease tumor growth. However, not all tumors respond well to anti-VEGF therapy. Despite much effort, identification of early response biomarkers that correlate with long-term efficacy of anti-VEGF therapy has been difficult. These difficulties arise in part because the functional effects of VEGF inhibition on tumor vessels are still unclear. We therefore assessed rapid molecular, morphologic and functional vascular responses following treatment with aflibercept (also known as VEGF Trap or ziv-aflibercept in the United States) in preclinical tumor models with a range of responses to anti-VEGF therapy, including Colo205 human colorectal carcinoma (highly sensitive), C6 rat glioblastoma (moderately sensitive), and HT1080 human fibrosarcoma (resistant), and correlated these changes to long-term tumor growth inhibition. We found that an overall decrease in tumor vessel perfusion, assessed by dynamic contrast-enhanced ultrasound (DCE-US), and increases in tumor hypoxia correlated well with long-term tumor growth inhibition, whereas changes in vascular gene expression and microvessel density did not. Our findings support previous clinical studies showing that decreased tumor perfusion after anti-VEGF therapy (measured by DCE-US) correlated with response. Thus, measuring tumor perfusion changes shortly after treatment with VEGF inhibitors, or possibly other anti-angiogenic therapies, may be useful to predict treatment efficacy.

The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes have not been previously developed. Here we show that dendritic cells from mice lacking the three immunoproteasome catalytic subunits display defects in presenting multiple major histocompatability (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes is markedly reduced in immunoproteasome-deficient animals, while presentation of MHC class II peptides is unaffected. By mass spectrometry the repertoire of MHC class I-presented peptides is ~50% different and these differences are sufficient to stimulate robust transplant rejection of wild type cells in mutant mice. These results indicate that immunoproteasomes play a much more important role in antigen presentation than previously thought.

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.

Electronic supplementary material

The online version of this article (doi:10.1007/s10456-011-9249-6) contains supplementary material, which is available to authorized users.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease thought to be mediated by dysfunctional innate and/or adaptive immunity. This aberrant immune response leads to the secretion of harmful cytokines that destroy the epithelium of the gastrointestinal tract leading to further inflammation. IL-22 is a Th17 T cell associated cytokine that is bi-functional with both pro-inflammatory and protective effects on tissues depending on the inflammatory context. We show herein that IL-22 protects mice from IBD. Interestingly, this protection is not only mediated by CD4 T cells, but IL-22 expressing NK cells also confer protection. In addition, IL-22 expression is differentially regulated between NK cell subsets. Thus, both the innate and adaptive immune responses have developed protective mechanisms to counteract the damaging effects of inflammation on tissues.

Approval of the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab by the FDA in 2004 reflected the success of this vascular targeting strategy in extending survival in patients with advanced cancers. However, consistent with previous reports that experimental tumors can grow or recur during VEGF blockade, it has become clear that many patients treated with VEGF inhibitors will ultimately develop progressive disease. Previous studies have shown that disruption of VEGF signaling in tumors induces remodeling in surviving vessels, and link increased expression of angiopoietin-1 (Ang-1) with this process. However, overexpression of Ang-1 in different tumors has yielded divergent results, restricting angiogenesis in some systems while promoting it in others. These data raise the possibility that effects of Ang-1/Tie-2 may be context-dependent. Expression of an Ang-1 construct (Ang1*) did not significantly change tumor growth in our model prior to treatment, although vessels exhibited changes consistent with increased Tie-2 signaling. During inhibition of VEGF, however, both overexpression of Ang1* and administration of an engineered Ang-1 agonist (Bow-Ang1) strikingly protected tumors and vasculature from regression. In this context, Ang-1/Tie-2 activation limited tumor hypoxia, increased vessel caliber, and promoted recruitment of mural cells. Thus, these studies support a model in which activation of Tie-2 is important for tumor and vessel survival when VEGF-dependent vasculature is stressed. Understanding such mechanisms of adaptation to this validated form of therapy may be important in designing regimens that make the best use of this approach.

The mechanism by which MSU crystals intracellularly activate the Cryopyrin inflammasome is unknown. Here, we used a mouse molecular genetics-based approach to test if the LRR domain of Cyopyrin is required for MSU crystal-induced inflammation.

Release of IL-1β, but not CXCL1 and TNFα, was impaired in CryoΔLRR Z/ΔLRR Z and Cryo−Z/−Z BMDMs compared to WT BMDMs in response to not only MSU crystals but also other known stumuli that activate the cryopyrin inflammasome. In addition, comparable percentage of MSU crystals taken up by each type of BMDMs was observed. Moreover, total leukocytes infiltrated in the air pouch and IL-1β production were attenuated in Cryo−Z/−Z and CryoΔLRR Z/ΔLRR Z mice at 6 h post-injection of MSU crystals compared to WT mice.

Conclusions

MSU crystal-induced inflammatory responses were comparably attenuated both in vitro and in vivo in CryoΔLRR Z/ΔLRR Z and Cryo−Z/−Z mice. Hence, the LRR domain of Cryopyrin plays a role in mediating MSU crystal-induced inflammation in this model.

A novel subretinal Matrigel model of choroidal neovascularization (CNV) was devised, with several unique features that mimic those in human exudative (wet) AMD. With this model and VEGF Trap, a potent receptor-based inhibitor of VEGF-A and PlGF, the data show that inhibition of VEGF-A, and perhaps PlGF as well, not only stops the growth and induces regression of experimental CNV, but also inhibits the associated inflammation and fibrotic responses.

Purpose.

The exudative, or the wet form of age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). A subretinal Matrigel (BD Biosciences, Bedford MA) model of CNV is described here, along with the effects of vascular endothelial growth factor (VEGF) neutralization on the development of CNV and associated inflammation and fibrosis.

Methods.

CNV was induced in adult Sprague-Dawley rats by subretinal injection of Matrigel. CNV growth and associated leukocyte infiltration and collagen deposition were examined. VEGF Trap (Regeneron Pharmaceuticals, Tarrytown, NY), a recombinant protein that comprises portions of the extracellular domains of VEGF receptors 1 and 2 and that binds all isoforms of VEGF-A as well as placental growth factor with high affinity, was administered subcutaneously.

Results.

Initiation of CNV was detected 4 days after Matrigel injection and then increased progressively in size. Systemic administration of VEGF Trap beginning on day 2 and 6 completely prevented development of CNV. When CNV was allowed to develop for 10 days before treatment was initiated, VEGF Trap not only prevented its further progression, but also induced substantial regression of existing lesions. In addition, VEGF Trap treatment reduced the total lesion volume and largely prevented the progressive leukocyte infiltration and fibrosis associated with CNV.

Conclusions.

The subretinal Matrigel CNV model provides a convenient tool for the study of the diverse components of complex CNV lesions. The data not only confirm the critical roles of VEGF in the development and maintenance of CNV, but further demonstrate that VEGF and other VEGF receptor 1 ligands promote CNV-associated inflammation and fibrosis.

Obsessive-compulsive disorder (OCD) is a common psychiatric disorder defined by the presence of obsessive thoughts and repetitive compulsive actions, and it often encompasses anxiety and depressive symptoms1,2. Recently, the corticostriatal circuitry has been implicated in the pathogenesis of OCD3,4. However, the etiology, pathophysiology and molecular basis of OCD remain unknown. Several studies indicate that the pathogenesis of OCD has a genetic component5–8. Here we demonstrate that loss of a neuron-specific transmembrane protein, SLIT and NTRK-like protein-5 (Slitrk5), leads to OCD-like behaviors in mice, which manifests as excessive self-grooming and increased anxiety-like behaviors, and is alleviated by the selective serotonin reuptake inhibitor fluoxetine. Slitrk5−/− mice show selective overactivation of the orbitofrontal cortex, abnormalities in striatal anatomy and cell morphology and alterations in glutamate receptor composition, which contribute to deficient corticostriatal neurotransmission. Thus, our studies identify Slitrk5 as an essential molecule at corticostriatal synapses and provide a new mouse model of OCD-like behaviors.

Blood vessel formation is controlled by the balance between pro- and anti-angiogenic pathways. Although much is known about the factors that drive sprouting of neovessels, the factors that stabilize and pattern neovessels are undefined. The expression of angiomodulin (AGM), a VEGF-A binding protein, was increased in the vasculature of several human tumors as compared to normal tissue, raising the hypothesis that AGM may modulate VEGF-A-dependent vascular patterning. To elucidate the expression pattern of AGM, we developed an AGM knockin reporter mouse (AGMlacZ/+) wherein we demonstrate that AGM is predominantly expressed in the vasculature of developing embryos and adult organs. During physiological and pathological angiogenesis, AGM is upregulated in the angiogenic vasculature. Using the zebrafish model, we found that AGM is restricted to developing vasculature by 17-22 hpf. Blockade of AGM activity with morpholino oligomers (MO) results in prominent angiogenesis defects in vascular sprouting and remodeling. Concurrent knockdown of both AGM and VEGF-A results in synergistic angiogenesis defects. When VEGF-A is overexpressed, the compensatory induction of the VEGF-A receptor, VEGFR-2/flk-1, is blocked by the simultaneous injection of AGM MO. These results demonstrate that the vascular-specific marker AGM modulates vascular remodeling in part by temporizing the pro-angiogenic effects of VEGF-A.

Adult mammalian testis is a source of pluripotent stem cells1. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells2. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro1, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multi-potent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125–β-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125–LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125–LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125− cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular book marking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

TLRs play an essential role in the induction of immune responses by detecting
conserved molecular products of microorganisms. However, the function of TLR8 is
largely unknown. In the current study, we investigated the role of TLR8 signaling in
immunity in mice. We found that
Tlr8–/– DCs overexpressed
TLR7, were hyperresponsive to various TLR7 ligands, and showed stronger and faster
NF-κB activation upon stimulation with the TLR7 ligand R848.
Tlr8–/– mice showed
splenomegaly, defective development of marginal zone (MZ) and B1 B cells, and
increased serum levels of IgM and IgG2a. Furthermore,
Tlr8–/– mice exhibited
increased serum levels of autoantibodies against small nuclear ribonucleoproteins,
ribonucleoprotein, and dsDNA and developed glomerulonephritis, whereas neither
Tlr7–/– nor
Tlr8–/–Tlr7–/–
mice showed any of the phenotypes observed in
Tlr8–/– mice. These data
provide evidence for a pivotal role for mouse TLR8 in the regulation of mouse TLR7
expression and prevention of spontaneous autoimmunity.

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.

Molecular mechanisms regulating the remodeling of the lymphatic vasculature from an immature plexus of vessels to a hierarchal network of initial and collecting lymphatics are not well understood. One gene thought to be important for this process is Angiopoietin-2 (Ang-2). Ang2-/- mice have previously been reported to exhibit an abnormal lymphatic phenotype but the precise nature of the lymphatic defects and the underlying mechanisms have yet to be defined. Here we demonstrate by whole-mount immunofluorescence staining of ear skin and mesentery that lymphatic vessels in Ang2-/- mice fail to mature and do not exhibit a collecting vessel phenotype. Furthermore, dermal lymphatic vessels in Ang2-/- pups prematurely recruit smooth muscle cells and do not undergo proper postnatal remodeling. In contrast, Ang2 knockout Ang1 knock-in mice do develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. Taken together, this work pinpoints a specific lymphatic defect of Ang2-/- mice and further defines the sequential steps in lymphatic vessel remodeling.

Hemangiomas are the most common type of tumor in infants. As they are endothelial cell–derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T–transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/–, and Ang2–/– mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/– cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/– cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.

The interleukin 4 receptor (IL-4R) is a central mediator of T helper type 2 (TH2)–mediated disease and associates with either the common γ-chain to form the type I IL-4R or with the IL-13R α1 chain (IL-13Rα1) to form the type II IL-4R. Here we used Il13ra1−/− mice to characterize the distinct functions of type I and type II IL-4 receptors in vivo. In contrast to Il4ra−/− mice, which have weak TH2 responses, Il13ra1−/− mice had exacerbated TH2 responses. Il13ra1−/− mice showed much less mortality after infection with Schistosoma mansoni and much more susceptibility to Nippostrongylus brasiliensis. IL-13Rα1 was essential for allergen-induced airway hyperreactivity and mucus hypersecretion but not for fibroblast or alternative macrophage activation. Thus, type I and II IL-4 receptors exert distinct effects on immune responses.

To examine the functional and therapeutic significance of pericytes in ovarian cancer vasculature.

Study Design

Tumor vessel morphology and efficacy of endothelial and pericyte targeting were examined using in vivo ovarian cancer models. The expression of platelet derived growth factor (PDGF) ligands and receptors was examined in endothelial, pericyte-like, and ovarian cancer cells.

Results

Relative to normal vessels, tumor vasculature was characterized by loosely attached pericytes in reduced density. PDGF-BB was expressed predominantly by the endothelial and cancer cells whereas PDGFRβ was present in pericyte-like cells. PDGF-BB significantly increased migration of and VEGF production by pericyte-like cells while PDGFRβ blockade abrogated these effects. Dual VEGF (VEGF-Trap) and PDGF-B (PDGF-Trap) targeted therapy was more effective in inhibiting in vivo tumor growth than either agent alone.

Conclusions

Aberrations in the tumor microenvironment contribute to endothelial cell survival. Strategies targeting both endothelial cells and pericytes should be considered for clinical trials.